Numerous methods and systems have been developed for the detection and quantitation of analytes of interest in biochemical and biological substances. Methods and systems which are capable of measuring trace amounts of microorganisms, pharmaceuticals, hormones, viruses, antibodies, nucleic acids and other proteins are of great value to researchers and clinicians.
A very substantial body of art has been developed based upon well known binding reactions, e.g., antigen-antibody reactions, nucleic acid hybridization techniques and protein-ligand systems. The high degree of specificity in many biochemical and biological binding systems has led to many assay methods and systems of value in research and diagnostics. Typically, the existence of an analyte of interest is indicated by the presence or absence of an observable "label" attached to one or more of the binding materials. Of particular interest are labels which can be made to luminesce through photochemical, chemical, and electrochemical means.
"Photoluminescence" is the process whereby a material is induced to luminesce when it absorbs electromagnetic radiation. Fluorescence and phosphorescence are types of photoluminescence. "Chemilumnescent" processes entail the creation of luminescent species by chemical transfer of energy. "Electrochemiluminescence" entails creation of luminescent species electrochemically.
Many chemiluminescent assay techniques, where a sample containing an analyte of interest is mixed with a reactant, labeled with a chemiluminescent label, have been developed. The reactive mixture is incubated and some portion of the labeled reactant binds to the analyte. After incubation, the bound and unbound fractions of the mixture are separated and the concentration of the label in either or both fractions can be determined by chemiluminescent techniques. The level of chemiluminescence determined in one or both fractions indicates the amount of analyte of interest in the biological sample.
Electrochemiluminescent (ECL) assaying techniques are an improvement over other assaying techniques. They provide a sensitive and precise measurement of the presence and concentration of an analyte of interest. In such techniques, the incubated sample is exposed to a voltametric working electrode in order to trigger luminescence. In the proper chemical environment, such electrochemiluminescence is triggered by a voltage impressed on the working electrode at a particular time and in a particular manner. The light produced by the label is measured and indicates the presence or quantity of the analyte. For a fuller description of such ECL techniques, reference is made to PCT published application Ser. No. 85/01253 (W086/02734), PCT published application Ser. No. 87/00987 (W087/06706) and PCT published application Ser. No. 88/03947 (W089/04302). The disclosures of the aforesaid applications are incorporated by reference.
Additionally, U.S. patent application Ser. No. 267,509 and U.S. patent application Ser. No. 266,914 relate to preferred assay compositions; U.S. Pat. No. 5,061,445 and U.S. patent application Ser. No. 744,890, teach preferred apparatus for conducting ECL-base assays; and U.S. patent application Ser. No. 652,427 describes preferred methods and apparatus for conducting ECL-based assays. The disclosure of these patents and applications are incorporated by reference as well.
The methods taught in PCT published application Ser. No. 89/04919 (WO90/05301) permit the detection and quantitation of extremely small quantities of analytes in a variety of assays performed in research and clinical settings. However, the demands of researchers and clinicians always make it imperative to try to lower the detection limits of assays performed by these methods, to increase the sensitivities of those assays and to increase the speed at which they can be performed.
In particular, the demand exists for improved DNA probe assays. In this regard, applicants have found that analytes of interest can be detected using specific labeled compounds. Some of these labeled compounds are known, for example, from AU 33343/89 and from Bannwarth et al., "A Simple Specific Labelling for Oligonucleotides by Bathophenanthroline . Ru.sup.II Complexes as Nonradioactive Label Molecules", Tetrahedron Letters, Vol. 30, No. 12, pp. 1513-1516, 1989, and others are new.